Regulation of Autolysis-Dependent Extracellular DNA Release by Enterococcus faecalis Extracellular Proteases Influences Biofilm Development

Abstract

Enterococci are major contributors of hospital-acquired infections and have emerged as important reservoirs for the dissemination of antibiotic resistance traits. The ability to form biofilms on medical devices is an important aspect of pathogenesis in the hospital environment. The Enterococcus faecalis Fsr quorum system has been shown to regulate biofilm formation through the production of gelatinase, but the mechanism has been hitherto unknown. Here we show that both gelatinase (GelE) and serine protease (SprE) contribute to biofilm formation by E. faecalis and provide clues to how the activity of these proteases governs this developmental process. Confocal imaging of biofilms suggested that GelE ؊ mutants were significantly reduced in biofilm biomass compared to the parental strain, whereas the absence of SprE appeared to accelerate the progression of biofilm development. The phenotype observed in a SprE ؊ mutant was linked to an observed increase in autolytic rate compared to the parental strain. Culture supernatant analysis and confocal microscopy confirmed the inability of mutants deficient in GelE to release extracellular DNA (eDNA) in planktonic and biofilm cultures, whereas cells deficient in SprE produced significantly more eDNA as a component of the biofilm matrix. DNase I treatment of E. faecalis biofilms reduced the accumulation of biofilm, implying a critical role for eDNA in biofilm development. In conclusion, our data suggest that the interplay of two secreted and coregulated proteases-GelE and SprE-is responsible for regulating autolysis and the release of highmolecular-weight eDNA, a critical component for the development of E. faecalis biofilms.

Phage-induced lysis enhances biofilm formation in Shewanella oneidensis MR-1

“…In Neisseria, DNA release is thought to be mediated by lytic transglycosylases and N-acetylmuramyl-L-alanine amidase . Other factors implicated in cell lysis are toxin/antitoxin system that have been characterized, for example, in Enterococcus faecalis (Thomas et al, 2008) and Staphylococcus sp. (Qin et al, 2007;Rice et al, 2007;Mann et al, 2009).…”

“…In contrast, the significance of eDNA for cellular attachment and structural integrity has more recently been recognized for an increasing number of Gram-negative and Grampositive species (Whitchurch et al, 2002;Steinberger and Holden, 2005;Allesen-Holm et al, 2006;Moscoso et al, 2006;Jurcisek and Bakaletz, 2007;Qin et al, 2007;Izano et al, 2008;Thomas et al, 2008;Heijstra et al, 2009;Vilain et al, 2009;Harmsen et al, 2010;Lappann et al, 2010). Release of DNA in bacterial biofilms has mainly been attributed to the lysis of a cellular subpopulation, mediated by the activity of autolysis systems (Allesen-Holm et al, 2006;Rice et al, 2007;Thomas et al, 2008Thomas et al, , 2009Mann et al, 2009). The analysis of aggregates formed by S. oneidensis MR-1 in planktonic cultures indicated the presence of proteins, a-D-mannose or a-D-glucose containing exopolysaccharides, and substantial amounts of eDNA (McLean et al, 2008b).…”

Inhibition of biofilm in Bacillus amyloliquefaciens Q-426 by diketopiperazines

“…Biofilms have been implicated in many persistent and chronic bacterial infections, including periodocarditis, endocarditis, osteomyelitis, caries, chronic lung infections in cystic fibrosis patients, urinary tract infections, chronic otitis media, gastrointestinal ulcers (Costerton et al 1999). The causative microorganisms of biofilm-associated infections are different among Grampositive species of Staphylococcus, Streptococcus (Karlowsky et al 2004) and Enterococcus (Thomas et al 2008) as well as Gram-negative bacteria, such as Pseudomonas aeruginosa (Purevdorj et al 2002), Escherichia coli, Actinobacillus actinomycetemcomitans (Kaplan et al 2004), and Burkholderia cenocepacia (Huber et al 2001).…”

NontypeableHaemophilus influenzaereleases DNA and DNABII proteins via a T4SS-like complex and ComE of the type IV pilus machinery

“…Members of the DNABII family of DNA-binding proteins, integration host factor (IHF) and histone-like protein (HU), are known for their strong structural influences on DNA intracellularly (18-24), and are also critical to the stability of the lattice-like 3D DNA structure found in biofilm matrices extracellularly (13,(25)(26)(27)(28)(29)(30)(31). To date, the presence of bacterial eDNA within biofilms has been attributed to release via various mechanisms, including cell lysis (autolysis or phagemediated) (12,32,33), or active secretion through type IV secretion systems (T4SSs) (34,35). Recently, DNA has been shown to be released from Pseudomonas aeruginosa by explosive cell lysis that is predicated on the formation of giant rounded cells that ultimately rapidly lyse, thereby releasing cell contents into the environment (36).…”

“…Extracellular DNA (eDNA) is a major constituent of the EPS formed by multiple human pathogens (5-7) and serves diverse roles within the bacterial biofilm. eDNA provides structural stability to the matrix, acts as a sink for antimicrobial peptides, protects resident bacteria from the host immune response, provides a source of "common goods" for the resident bacteria, acts as a universal structural material conducive to microbial community architecture, and facilitates the uptake of genetic material between bacterial species via a process known as horizontal gene transfer (5,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Members of the DNABII family of DNA-binding proteins, integration host factor (IHF) and histone-like protein (HU), are known for their strong structural influences on DNA intracellularly (18)(19)(20)(21)(22)(23)(24), and are also critical to the stability of the lattice-like 3D DNA structure found in biofilm matrices extracellularly (13,(25)(26)(27)(28)(29)(30)(31).…”